The members of the PII (GlnB) protein family are among the most broadly distributed regulatory proteins and are primarily involved in central nitrogen regulation in bacteria, archaea and in some plants. This protein family is biologically important not only for its distribution, but also for the critical roles that it has been shown to play in all aspects of nitrogen regulation and the balancing of carbon and nitrogen utilization. In the past few years, three very significant developments have occurred that this proposal seeks to take advantage of: The crystal structures of two members of this family have been solved, many organisms have been shown to have more than one homolog, and homologs in different organisms have been implicated in rather different biochemical functions. A critical issue, and a focus of this proposal, is the molecular basis of these different biochemical roles. This proposal will address this issue using our recent observation that Rhodospirillum rubrum has three homologs (named GlnB, GlnJ, and GlnK), with rather similar sequences, but with at least four different readily assayed functions in vivo. This provides an excellent model system for identifying functionally important residues in these homologs, correlating them with specific biological roles and eventually determining the specific receptor proteins that interact with the PII (GlnB) homologs in these roles. Identification of the critical regions of the PII homologs involved in different protein interactions will be of biological importance, especially when coupled with a better understanding of the proteins with which they interact. We cannot understand the diverse metabolic roles of the PII family until we have precisely this information. Only such information will, for example, explain the role of the post-translational uridylylation that is common among PII homologs. Given the extreme conservation of PII sequence across much of biology, it is a reasonable hypothesis that critical surfaces involved in the interactions of the PII homologs of R. rubrum will serve as important regions in other PII homologs as well